Biochemical evidence for autocrine/paracrine regulation of apoptosis in cultured uterine epithelial cells during mouse embryo implantation in vitro

Abstract

During embryo implantation, apoptosis is observed morphologically at the implantation site of endometrium. The objectives of this study were to demonstrate biochemical evidence of apoptosis and quantitative assessment of DNA fragmentation in uterine epithelial cells using a mouse implantation model, and to investigate the autocrine/paracrine regulation of apoptosis in uterine epithelial cells during blastocyst outgrowth. Blastocysts from day 4 pregnant mice were cultured on uterine epithelial cells for 96 h. Uterine epithelial cells dislodged by trophoblasts in endometrium-trophoblast unit demonstrated morphological features of apoptosis by Acridine Orange staining. Electrophoresis demonstrated DNA ladder and DNA fragmentation by enzyme- linked immunosorbent assay markedly increased after 48 h period of incubation. Apoptosis increased in an exponential way in accordance with trophoblast outgrowth. In addition, DNA fragmentation was shown in the epithelial cells by adding embryo-conditioned medium (CM) and the effect of embryo CM on apoptosis was significantly inhibited by anti-transforming growth factor (TGF)-β antibody. Delayed outgrowth was observed after 48 h of incubation in the blastocysts cultured with anti-TGF-β antibody. These results suggest there is autocrine/paracrine regulation of apoptosis in uterine epithelial cells at mouse embryo implantation and that TGF-β might play an important role in the occurrence of apoptosis in the endometrium- trophoblast unit.